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Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates

2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês

10.1016/j.jbc.2022.102522

1083-351X

F Kuhlmann, Phillip Key, Suzanne M. Hickerson, John Turk, Fong‐Fu Hsu, Stephen M. Beverley,

Lysosomal Storage Disorders Research

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs−). Unexpectedly and unlike fungi where IPCS is essential, Δipcs− was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs− promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs−, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs− parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis. Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs−). Unexpectedly and unlike fungi where IPCS is essential, Δipcs− was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs− promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs−, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs− parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis. Leishmaniasis is often considered a neglected tropical disease. Currently, it is estimated that there are more than 1.7 billion people at risk and nearly 12 million people with symptomatic disease, ranging from mild cutaneous infections to severe disfiguring or lethal forms, with upward of 50,000 deaths annually (1Alvar J. Velez I.D. Bern C. Herrero M. Desjeux P. Cano J. et al.Leishmaniasis worldwide and global estimates of its incidence.PLoS One. 2012; 7e35671Google Scholar, 2Pigott D.M. Bhatt S. Golding N. Duda K.A. Battle K.E. Brady O.J. et al.Global distribution maps of the leishmaniases.Elife. 2014; 3e28051Google Scholar, 3Banuls A.L. Bastien P. Pomares C. Arevalo J. Fisa R. Hide M. Clinical pleiomorphism in human leishmaniases, with special mention of asymptomatic infection.Clin. Microbiol. Infect. 2011; 17: 1451-1461Google Scholar, 4Herwaldt B.L. Leishmaniasis.Lancet. 1999; 354: 1191-1199Google Scholar). Remarkably, the great majority of infections are undiagnosed and/or asymptomatic, suggesting that well more than 100 million people may harbor parasites (3Banuls A.L. Bastien P. Pomares C. Arevalo J. Fisa R. Hide M. Clinical pleiomorphism in human leishmaniases, with special mention of asymptomatic infection.Clin. Microbiol. Infect. 2011; 17: 1451-1461Google Scholar, 5Mannan S.B. Elhadad H. Loc T.T.H. Sadik M. Mohamed M.Y.F. Nam N.H. et al.Prevalence and associated factors of asymptomatic leishmaniasis: a systematic review and meta-analysis.Parasitol. Int. 2021; 81102229Google Scholar). Persistent asymptomatic infections constitute a double edged sword; while serving as a reservoir for transmission and/or reactivation by stress or immunosuppression, persistent parasites are known to mediate strong protective concomitant immunity against disease pathology (3Banuls A.L. Bastien P. Pomares C. Arevalo J. Fisa R. Hide M. Clinical pleiomorphism in human leishmaniases, with special mention of asymptomatic infection.Clin. Microbiol. Infect. 2011; 17: 1451-1461Google Scholar, 5Mannan S.B. Elhadad H. Loc T.T.H. Sadik M. Mohamed M.Y.F. Nam N.H. et al.Prevalence and associated factors of asymptomatic leishmaniasis: a systematic review and meta-analysis.Parasitol. Int. 2021; 81102229Google Scholar, 6WHOControl of the leishmaniases: WHO expert committee on the control of leishmaniases.World Health Organ. Tech. Rep. 2010; 949: 1-186Google Scholar, 7Singh O.P. Hasker E. Sacks D. Boelaert M. Sundar S. Asymptomatic Leishmania infection: a new challenge for Leishmania control.Clin. Infect. Dis. 2014; 58: 1424-1429Google Scholar). Leishmania have two distinct growth stages, a promastigote stage in the sand fly vector and an intracellular amastigote stage residing within cellular endocytic pathways in the mammalian host (4Herwaldt B.L. Leishmaniasis.Lancet. 1999; 354: 1191-1199Google Scholar). While the promastigote stage is readily cultured in the laboratory and amenable to molecular techniques, amastigotes require the use of macrophage infection systems or infections of animal models replicating key aspects of human disease. While progress has been achieved in new chemotherapeutic strategies, many are not yet implemented clinically and resistance to several promising agents has already been observed (8Croft S.L. Sundar S. Fairlamb A.H. Drug resistance in leishmaniasis.Clin. Microbiol. Rev. 2006; 19: 111-126Google Scholar, 9den Boer M. Argaw D. Jannin J. Alvar J. Leishmaniasis impact and treatment access.Clin. Microbiol. Infect. 2011; 17: 1471-1477Google Scholar, 10Sangshetti J.N. Khan F.A.K. Kulkarni A.K. Arote R. Patil R.H. Antileishmanial drug discovery: comprehensive review of the last 10 years.R. Soc. Chem. 2015; 5: 32376-32415Google Scholar). One pathway potentially offering important drug targets is that of sphingolipid (SL) synthesis (11Zhang K. Bangs J.D. Beverley S.M. Sphingolipids in parasitic protozoa.Adv. Exp. Med. Biol. 2010; 688: 238-248Google Scholar, 12Mina J.G.M. Denny P.W. Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis.Parasitology. 2018; 145: 134-147Google Scholar, 13Guan X.L. Maser P. Comparative sphingolipidomics of disease-causing trypanosomatids reveal unique lifecycle- and taxonomy-specific lipid chemistries.Sci. Rep. 2017; 713617Google Scholar). SLs are a diverse class of lipids that function in apoptosis, cell signaling, and membrane structure, and these pathways have been targeted for therapies against a wide range of diseases including cancer, multiple sclerosis, and infectious diseases (14Oskouian B. Saba J.D. Cancer treatment strategies targeting sphingolipid metabolism.Adv. Exp. Med. Biol. 2010; 688: 185-205Google Scholar, 15Koeller C.M. Heise N. The sphingolipid biosynthetic pathway is a potential target for chemotherapy against chagas disease.Enzyme Res. 2011; 2011648159Google Scholar, 16Suzuki E. Tanaka A.K. Toledo M.S. Levery S.B. Straus A.H. Takahashi H.K. Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies.Biochim. Biophys. Acta. 2008; 1780: 362-369Google Scholar, 17Hannun Y.A. Obeid L.M. Sphingolipids and their metabolism in physiology and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 175-191Google Scholar, 18Megyeri M. Riezman H. Schuldiner M. Futerman A.H. Making sense of the yeast sphingolipid pathway.J. Mol. Biol. 2016; 428: 4765-4775Google Scholar). SLs differ in the nature of the head groups, as well as the composition of the hydrophobic ceramide anchor, in different tissues and species. Unlike mammalian cells that primarily synthesize sphingomyelin (SM) and complex glycosphingolipids, Leishmania synthesize inositol phosphorylceramide (IPC) as their primary SL (19Kaneshiro E.S. Jayasimhulu K. Lester R.L. Characterization of inositol lipids from Leishmania donovani promastigotes: identification of an inositol sphingophospholipid.J. Lipid Res. 1986; 27: 1294-1303Google Scholar), as do other protozoa, fungi, and plants (11Zhang K. Bangs J.D. Beverley S.M. Sphingolipids in parasitic protozoa.Adv. Exp. Med. Biol. 2010; 688: 238-248Google Scholar, 20Harrison P.J. Dunn T.M. Campopiano D.J. Sphingolipid biosynthesis in man and microbes.Nat. Prod. Rep. 2018; 35: 921-954Google Scholar, 21Pinneh E.C. Stoppel R. Knight H. Knight M.R. Steel P.G. Denny P.W. Expression levels of inositol phosphorylceramide synthase modulate plant responses to biotic and abiotic stress in Arabidopsis thaliana.PLoS One. 2019; 14e0217087Google Scholar). This divergence has prompted scrutiny of IPC synthase (IPCS) as a drug target, where in fungi it is essential and repression causes decreased growth and virulence along with increased susceptibility to acidic environments (22Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. et al.Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans.Genes Dev. 2001; 15: 201-212Google Scholar, 23Heidler S.A. Radding J.A. Inositol phosphoryl transferases from human pathogenic fungi.Biochim. Biophys. Acta. 2000; 1500: 147-152Google Scholar). IPC is formed when IPCS converts ceramide and phosphatidylinositol (PI) to IPC and diacylglycerol (DAG). The related trypanosomatid parasite Trypanosoma brucei encodes four SL synthases (SLSs), collectively mediating synthesis of SM, ethanolamine (EtN) phosphorylceramide, and IPC; inducible RNAi knockdowns of the entire locus were lethal, although the contribution of the individual genes/activities was not further dissected (24Sutterwala S.S. Hsu F.F. Sevova E.S. Schwartz K.J. Zhang K. Key P. et al.Developmentally regulated sphingolipid synthesis in African trypanosomes.Mol. Microbiol. 2008; 70: 281-296Google Scholar). Leishmania IPCS has been pursued as a drug target by analogy of findings from pathogenic fungi as well as its divergence from mammalian SM synthases (25Denny P.W. Shams-Eldin H. Price H.P. Smith D.F. Schwarz R.T. The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase.J. Biol. Chem. 2006; 281: 28200-28209Google Scholar). Aureobasidin A, a known inhibitor of yeast IPCS, inhibits parasite growth at high concentrations, probably off-target from IPCS inhibition (25Denny P.W. Shams-Eldin H. Price H.P. Smith D.F. Schwarz R.T. The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase.J. Biol. Chem. 2006; 281: 28200-28209Google Scholar), but other candidate inhibitors have been advanced (26Sevova E.S. Goren M.A. Schwartz K.J. Hsu F.F. Turk J. Fox B.G. et al.Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa.J. Biol. Chem. 2010; 285: 20580-20587Google Scholar, 27Figueiredo J.M. Dias W.B. Mendonca-Previato L. Previato J.O. Heise N. Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi.Biochem. J. 2005; 387: 519-529Google Scholar, 28Mina J.G. Mosely J.A. Ali H.Z. Denny P.W. Steel P.G. Exploring Leishmania major inositol phosphorylceramide synthase (LmjIPCS): insights into the ceramide binding domain.Org. Biomol. Chem. 2011; 9: 1823-1830Google Scholar, 29Mina J.G. Mosely J.A. Ali H.Z. Shams-Eldin H. Schwarz R.T. Steel P.G. et al.A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase.Int. J. Biochem. Cell Biol. 2010; 42: 1553-1561Google Scholar, 30Norcliffe J.L. Mina J.G. Alvarez E. Cantizani J. de Dios-Anton F. Colmenarejo G. et al.Identifying inhibitors of the Leishmania inositol phosphorylceramide synthase with antiprotozoal activity using a yeast-based assay and ultra-high throughput screening platform.Sci. Rep. 2018; 8: 3938Google Scholar). Notably, the biological requirement for Leishmania IPCS has not been addressed in vivo. That Leishmania could differ from other organisms was first suggested by the fact that Leishmania major null mutants lacking the first enzyme in de novo SL synthesis, serine palmitoyl transferase (Δspt2-) completely lacked all SLs, yet remain viable in log phase culture (31Denny P.W. Goulding D. Ferguson M.A. Smith D.F. Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity.Mol. Microbiol. 2004; 52: 313-327Google Scholar, 32Zhang K. Showalter M. Revollo J. Hsu F.F. Turk J. Beverley S.M. Sphingolipids are essential for differentiation but not growth in Leishmania.EMBO J. 2003; 22: 6016-6026Google Scholar). Subsequent studies in promastigotes showed that SL catabolism was used to generate EtN, whose provision to Δspt2- parasites rescued a defect in differentiation to infective metacyclic parasites (33Zhang K. Pompey J.M. Hsu F.F. Key P. Bandhuvula P. Saba J.D. et al.Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania.EMBO J. 2007; 26: 1094-1104Google Scholar). While Δspt2- was fully infective in animal models, it continued to synthesize IPC, presumably by salvage of potential precursor SL components from the host cell (34Zhang K. Hsu F.F. Scott D.A. Docampo R. Turk J. Beverley S.M. Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis.Mol. Microbiol. 2005; 55: 1566-1578Google Scholar, 35Ali H.Z. Harding C.R. Denny P.W. Endocytosis and sphingolipid scavenging in Leishmania mexicana amastigotes.Biochem. Res. Int. 2011; 2012691363Google Scholar, 36Winter G. Fuchs M. McConville M.J. Stierhof Y.D. Overath P. Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid.J. Cell Sci. 1994; 107: 2471-2482Google Scholar). Several routes could be envisaged; sphingoid bases (SBs, also called long-chain bases) or ceramide precursors could be salvaged directly or mammalian SLs could be acquired and catabolized for resynthesis as IPC SLs. As all routes ultimately require IPCS for parasite IPC synthesis, we sought to generate Δipcs− null mutants if possible and to explore their impact on IPC synthesis and/or metabolism and virulence in animal models. Surprisingly, our data show convincingly IPCS was not essential, with SM salvage in WT cells rendering it the dominant amastigote SL as well as serving as the source of amastigote IPC. These findings likely account for the dispensability of IPCS in animal infections, potentially dampening enthusiasm for IPCS as a useful drug target in this parasite. The L. major genome is primarily diploid albeit with occasional aneuploidy, and typically two rounds of gene replacement are required to generate null mutants of single copy genes (37Cruz A. Coburn C.M. Beverley S.M. Double targeted gene replacement for creating null mutants.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Google Scholar). Targeting constructs containing resistance markers to G418 (NEO) or nourseothricin (SAT) were designed to successively replace the IPCS ORF precisely by homologous gene replacement (Fig. 1A). To alleviate concerns about lethality of IPCS deficiency, we focused on the culturable promastigote stage where SLs are not required in any capacity in the presence of EtN (31Denny P.W. Goulding D. Ferguson M.A. Smith D.F. Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity.Mol. Microbiol. 2004; 52: 313-327Google Scholar, 32Zhang K. Showalter M. Revollo J. Hsu F.F. Turk J. Beverley S.M. Sphingolipids are essential for differentiation but not growth in Leishmania.EMBO J. 2003; 22: 6016-6026Google Scholar, 33Zhang K. Pompey J.M. Hsu F.F. Key P. Bandhuvula P. Saba J.D. et al.Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania.EMBO J. 2007; 26: 1094-1104Google Scholar). A secondary effect of IPCS deficiency could be ceramide accumulation, which can be toxic (38Hannun Y.A. Obeid L.M. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind.J. Biol. Chem. 2002; 277: 25847-25850Google Scholar), and we also performed transfections in the presence of myriocin (an inhibitor of de novo SL synthesis) to potentially ameliorate this. Heterozygous (IPCS/IPCS::ΔNEO) and homozygous null mutants (IPCS::ΔSAT/IPCS::ΔNEO; referred to as Δipcs−) were readily obtained and confirmed by PCR tests to lack the IPCS coding region, accompanied by the expected planned replacements (analysis of the double homozygous replacement is shown in Fig. 1B). An episomal expression vector (pXG-BSD-IPCS) was used to restore IPCS expression, yielding the line Δipcs−/+IPCS (Fig. 1, A and B). The recovery of Δipcs− clonal lines was readily accomplished with good efficiencies, regardless of EtN or myriocin treatment, and these showed no strong growth defects. Numerous transfectant lines were obtained and authenticated with similar phenotypes, and in this work, results from one representative set of KO and IPCS-restored pair are shown. Enzymatic assays with promastigote microsomes were performed by evaluating the conversion of PI and 6-((N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)amino)hexanoyl)Sphingosine (NBD-Cer) to NBD-IPC, after TLC separation and quantitative fluoroscopy (Figs. 2A and S1A). High levels of NBD-IPC were generated in complete reactions bearing microsomes, NBD-Cer, and PI, and this product was sensitive to PI-PLC digestion as expected. We purified and confirmed the presumptive NBD-IPC from the TLC plate by mass spectrometry (MS) 1Data not shown.. Consistent with prior studies, addition of 20 μM aureobasidin A showed little inhibition of the in vitro reaction (26Sevova E.S. Goren M.A. Schwartz K.J. Hsu F.F. Turk J. Fox B.G. et al.Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa.J. Biol. Chem. 2010; 285: 20580-20587Google Scholar). Little or no product was obtained if PI or NBD-Cer were omitted, if phosphatidylcholine (PC) replaced PI or if microsomes were omitted or previously heat inactivated. These data confirmed our ability to assay IPCS, with the predicted enzymatic properties (Figs. 2A and S2A). We compared the IPCS activity of WT, Δipcs−, or Δipcs−/+IPCS parasite microsomes (Figs. 2B and S1B). As expected, Δipcs− showed no detectable activity, while Δipcs−/+IPCS showed about 50% more than WT (p < 0.001). MS was used to evaluate lipids present in whole cell extracts of WT, Δipcs−, and Δipcs−/+IPCS promastigotes (Fig. 3, A–C). In WT, two ion peaks at m/z 778.6 and 806.6 corresponding to [M – H]- ions of d16:1/18:0- and d18:1/18:0–IPC, respectively, were seen, as described previously (32Zhang K. Showalter M. Revollo J. Hsu F.F. Turk J. Beverley S.M. Sphingolipids are essential for differentiation but not growth in Leishmania.EMBO J. 2003; 22: 6016-6026Google Scholar, 34Zhang K. Hsu F.F. Scott D.A. Docampo R. Turk J. Beverley S.M. Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis.Mol. Microbiol. 2005; 55: 1566-1578Google Scholar, 39Hsu F.F. Turk J. Zhang K. Beverley S.M. Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2007; 18: 1591-1604Google Scholar). Consistent with the genetic deletion of IPCS, these IPC ions were undetectable in Δipcs− promastigotes. IPC levels were restored in the Δipcs−/+IPCS promastigotes, albeit only partially to about 50% WT levels (Fig. 3C). Together with the results from enzymatic assays, these data established that Leishmania IPCS is the sole source of IPCS enzymatic activity or IPC. As predicted, IPC precursor d16:1/18:0-ceramide (m/z 536.6) levels were elevated in the Δipcs− promastigotes (Fig. 3B) relative to WT cells (Fig. 3A). Curiously, this ceramide peak did not decrease noticeably in Δipcs−/+IPCS, despite restoration of IPC levels (Fig. 3C). Small variations in the levels of phosphatidylethanolamine (PE) ions of m/z 726.6 (p18:0/18:2-PE) and 728.6 (p18:0/18:1-PE) were seen, but the levels of other lipids identified by MS such as ions at m/z 849 (e18:0/18:1-PI) and 863 (18:0/18:1-PI) did not appear to change in either Δipcs− or Δipcs−/+IPCS promastigotes (Fig. 3, A–C). The causes of these minor changes were not pursued further. As promastigotes in vitro, Δipcs− grew somewhat slower than WT, with a doubling time of 9.0 ± 0.7 versus 7.7 ± 0.4 h for WT (mean ± 1 SD, n = 3), reaching a stationary phase density of about 70% WT (p < 0.05; Fig. 4A). Upon entry into stationary phase, a small percentage of nonviable cells emerged, as judged by propidium iodide exclusion (Fig. 4B). Since Δipcs− promastigotes should retain SLs capable of generating EtN (33Zhang K. Pompey J.M. Hsu F.F. Key P. Bandhuvula P. Saba J.D. et al.Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania.EMBO J. 2007; 26: 1094-1104Google Scholar), we suspected that provision of EtN would have no effect on stationary phase viability, which proved correct (Fig. S2, A and B). Restoration of IPCS expression in the Δipcs−/+IPCS line returned growth and viability to WT (Fig. 4, A and B). Upon entry into stationary phase, Leishmania promastigotes differentiate to an infective metacyclic form that can be purified by lectin or density gradient methods (40Spath G.F. Beverley S.M. A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.Exp. Parasitol. 2001; 99: 97-103Google Scholar). Δipcs− showed a small reduction in metacyclics relative to WT or the Δipcs−/+IPCS control (5.7 ± 2.7 % in Δipcs− versus 11.4 ± 7.1 % in WT or 10.4 ± 5.1% in Δipcs−/+IPCS; mean ± SD, n = 2; Fig. S2C; these differences were not statistically significant). Accumulation of ceramide, SBs, or their phosphorylated forms is toxic to most eukaryotic cells (38Hannun Y.A. Obeid L.M. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind.J. Biol. Chem. 2002; 277: 25847-25850Google Scholar). This suggested that in the absence of further metabolism by IPCS, addition of exogenous SBs would likely lead to excess toxic ceramide or SB levels in Δipcs−. Accordingly, daily addition of 2 μM 3-keto-dihydrosphingosine, dihydrosphingosine, or C17 phytosphingosine resulted in death of Δipcs− parasites (Fig. 4C), which was rescued in Δipcs−/+IPCS. For cultures receiving C17 phytosphingosine, MS confirmed synthesis of phytoceramide (Fig. S3). IPCS deficient Cryptococcus neoformans is susceptible to pH but not temperature stress (22Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. et al.Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans.Genes Dev. 2001; 15: 201-212Google Scholar), and we obtained similar results for L. major Δipcs− promastigotes. At pH 4.0, the WT, Δipcs−, and Δipcs−/+IPCS lines all died, while at pH 5.0, Δipcs− grew much slower than WT or Δipcs−/+IPCS (Fig. 4D). At pH 5.5 and above, no significant differences were seen amongst the three lines (Table S2). Temperature stress was tested at 30 °C and 33 °C with no significant effects on the relative growth of WT, Δipcs−, and Δipcs−/+IPCS lines (Fig. S2D); at 37 °C, all cells died. The IPCS product DAG also functions as a messenger in multiple signaling pathways mediated by PKC. Leishmania lack a known PKC, although it has been suggested that other kinases may fulfill similar roles (41Subramanya S. Mensa-Wilmot K. Diacylglycerol-stimulated endocytosis of transferrin in trypanosomatids is dependent on tyrosine kinase activity.PLoS One. 2010; 5e8538Google Scholar). We tested the ability of exogenous DAG to rescue the growth or viability reductions seen in Δipcs− promastigotes by adding DAG daily to cultures. However, with two separate supplementations (0.2 or 2 μM), no improvement was observed (Fig. S2, E and F), suggesting that DAG deficiency did not contribute to Δipcs− phenotypes. While we did not confirm DAG uptake by parasites here, that exogenous DAG is accessible to Leishmania was shown previously, as a brief 30 min exposure to 250 nM DAGs resulted in stimulation of transferrin endocytosis (41Subramanya S. Mensa-Wilmot K. Diacylglycerol-stimulated endocytosis of transferrin in trypanosomatids is dependent on tyrosine kinase activity.PLoS One. 2010; 5e8538Google Scholar). In Δspt2− promastigotes, SL deficiency causes changes in cell rounding, acidocalcisomes, and lipid bodies (33Zhang K. Pompey J.M. Hsu F.F. Key P. Bandhuvula P. Saba J.D. et al.Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania.EMBO J. 2007; 26: 1094-1104Google Scholar, 34Zhang K. Hsu F.F. Scott D.A. Docampo R. Turk J. Beverley S.M. Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis.Mol. Microbiol. 2005; 55: 1566-1578Google Scholar). In Δipcs−, a small increase in rounded cells was observed (21.6 ± 0.6% versus 7.4 ± 2.0% for WT or 8.9 ± 3.0% for Δipcs−/+IPCS, 2 replicates; p < 0.02 for comparisons of the mutant versus the other two lines). WT and Δipcs− appeared similar by 4′,6-diamidino-2-phenylindole staining of acidocalcisomes/polyphosphates or Nile Red O staining for lipid accumulation and lipid bodies (Fig. S4). To test the key question of whether IPCS is essential for virulence, we infected susceptible BALB/c or γ-interferon KO C57BL/6 mice with 1 × 105 metacyclic promastigotes. Unexpectedly, the Δipcs− infections progressed significantly faster than those of WT or Δipcs−/+IPCS, which were similar (a representative experiment is shown in Fig. 5 and combined data from four experiments are shown in Fig. S5A). Limiting dilution assays at key time points showed that regardless of genotype, lesions of similar sizes had similar parasite numbers (Fig. S5B), establishing that increased lesion size reflected increased parasite numbers primarily. These data show that if anything, Δipcs− was unexpectedly somewhat hypervirulent. To eliminate the possibility that amastigotes elaborated an alternative IPCS activity, parasites were inoculated into susceptible mice and purified from similarly sized lesions after 1 to 2 months. Lipids were then extracted for analysis by electrospray ionization MS (ESI/MS) in the negative ion mode. High amounts of background signal complicated interpretations of full scan spectra (Fig. S6), which was overcome by employing linked scan spectra using precursor ion scan of m/z 241 specific for detection of [M – H]- ions of PI and IPC lipids (39Hsu F.F. Turk J. Zhang K. Beverley S.M. Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2007; 18: 1591-1604Google Scholar, 42Hsu F.F. Turk J. Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study.J. Am. Soc. Mass Spectrom. 2000; 11: 986-999Google Scholar). These scans showed abundant IPC species (ions of m/z 778.6, 806.6, and 808.6) in purified WT amastigotes, as seen previously (Figs. 6A and S7, A and G) (34Zhang K. Hsu F.F. Scott D.A. Docampo R. Turk J. Beverley S.M. Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis.Mol. Microbiol. 2005; 55: 1566-1578Google Scholar). In Δipcs− amastigotes, only trace signals in the relevant m/z regions spanning the IPC m/z peaks of 778, 806, and 808 were evident, in four separate purified preparations (Figs. 6B and S1, C, E, H, and I). These peaks were considered background, first by virtue of their low signal/noise ratio (values <3 are considered noise and <10 insufficient for quantitation (43Currie L.A. Nomenclature in evaluation of analytical methods including detection and quantification capabilities (IUPAC Recommendations 1995).Pure Appl. Chem. 1995; 67: 1699-1723Google Scholar)) and secondly, a lack of the expected natural isotope pattern for real peaks (44Smith R.M. Understanding Mass Spectra. A Basic Approach.2nd ed. John Wiley & Sons, Inc., Hoboken, New Jersey2004Google Scholar). In contrast, these criteria validate the assigned IPC peaks in the WT purified amastigotes or infected tissues. The abundance of other lipid species did not appear to change consistently or significantly (Figs. S6–S8). No significant differences were seen in amastigotes purified from BALB/c or γ-IFN KO (γKO) C57BL/6 mice (Figs. S6–S8)